Targeting USP2 regulation of VPRBP-mediated degradation of p53 and PD-L1 for cancer therapy

Since Mdm2 (Mouse double minute 2) inhibitors show serious toxicity in clinic studies, different approaches to achieve therapeutic reactivation of p53-mediated tumor suppression in cancers need to be explored. Here, we identify the USP2 (ubiquitin specific peptidase 2)-VPRBP (viral protein R binding protein) axis as an important pathway for p53 regulation. Like Mdm2, VPRBP is a potent repressor of p53 but VPRBP stability is controlled by USP2. Interestingly, the USP2-VPRBP axis also regulates PD-L1 (programmed death-ligand 1) expression. Strikingly, the combination of a small-molecule USP2 inhibitor and anti-PD1 monoclonal antibody leads to complete regression of the tumors expressing wild-type p53. In contrast to Mdm2, knockout of Usp2 in mice has no obvious effect in normal tissues. Moreover, no obvious toxicity is observed upon the USP2 inhibitor treatment in vivo as Mdm2-mediated regulation of p53 remains intact. Our study reveals a promising strategy for p53-based therapy by circumventing the toxicity issue.


Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
The mass spectrometry data generated in this study have been deposited in the PRIDE (Proteomics IDEntifications Database) under accession code PXD040473 and PXD040477. The remaining data are available within the article and Supplementary Information. Source data are provided with this paper.
n/a n/a n/a n/a Sample sizes were determined to allow the statistical significance and based on the previous studies in the field (Zhang, J., Bu, X., Wang, H. et al. Nature 571, E10 2017;Mezzadra, R., Sun, C., Jae, L. et al.Nature 549, 106-110 2017;Lim S., Li C. et al. Cancer Cell 30, 925-939 2016. ). Sample size of each experiment was provided in figure legends.
No data was excluded from this study.
All experiments were repeated independently at least twice with similar results.
Samples were divided into each group randomly in all experiments.
Investigator was blinded to group allocation and data collection in in vivo experiments. Automated quantitative methods were used for IHC quantification to eliminate subjective interpretation of data. Blinding is not applicable for in vitro experiments in this study, because the same investigator performed cell culture, drug treatment and data analysis. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots. All cell lines were not authenticated.
The cell lines were tested negative for mycoplasma contamination No cell line used in the study was found in the databases of commonly misidentified cell lines that are maintained by ICLAC.
No wild animals were used in this study.
female nu/nu mice and Balb/c mice were used for EMT6 mouse models. Male C57BL/6J mice were used for RM-1 mouse model.
No field-collected samples were used in this study.
This study is compliant with the relevant ethical regulations for animal experiments. All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC)of Columbia University .
Tissues were dissected from mice and digested with Collagase at 37C for 1h. Single cell suspension were prepared with 40uM cell strainer, then subjected to staining with specific antibodies and flow cytometry.

Flowjo V10
at least 10000 cells were counted in each samples.